DICER1 platform domain missense variants inhibit miRNA biogenesis and lead to tumor susceptibility

Abstract The endoribonuclease DICER1 plays an essential role in the microRNA (miRNA) biogenesis pathway, cleaving precursor miRNA (pre-miRNA) stem-loops to generate mature single-stranded miRNAs. Germline pathogenic variants (GPVs) in DICER1 result in DICER1 tumor predisposition syndrome (DTPS), a mainly childhood-onset tumor susceptibility disorder. Most DTPS-causing GPVs are nonsense or frameshifting, with tumor development requiring a second somatic missense hit that impairs the DICER1 RNase IIIb domain. Interestingly, germline DICER1 missense variants that cluster in the DICER1 Platform domain have been identified in some persons affected by tumors that also associate with DTPS. Here, we demonstrate that four of these Platform domain variants prevent DICER1 from producing mature miRNAs and as a result impair miRNA-mediated gene silencing. Importantly, we show that in contrast to canonical somatic missense variants that alter DICER1 cleavage activity, DICER1 proteins harboring these Platform variants fail to bind to pre-miRNA stem-loops. Taken together, this work sheds light upon a unique subset of GPVs causing DTPS and provides new insights into how alterations in the DICER1 Platform domain can impact miRNA biogenesis.

an important role in providing distance between domains involved in pre-miRNA binding (Platform and PAZ) and pre-miRNA cleavage (RNase IIIa and IIIb). As a result, this helix allows DICER1 to cleave pre-miRNA at around 22 nucleotides from both its 3 and 5 ends ( 9 , 10 , 1 , 2 ) ( 6 , 7 ). This cleavage e v ent is catalyzed by the RNase IIIa and RNase IIIb domains ( 1 , 3 ). These domains form a heterodimer around the pre-miRNA stem to mediate two cleavage e v ents catalyzed by ion binding residues using magnesium or manganese as a co-factor. Dicer RNase IIIa and RNase IIIb domains cleave pre-miRNA stem-loops to generate mature 3 arm (3p) and 5 arm (5p) miRNAs, respecti v ely ( 1 , 3 ). Finally, the C-terminus of the protein contains a doublestranded RNA-binding domain (dsRBD) that plays a role in pre-miRNA binding and clamping the RNA in the Dicer catalytic sites ( 5 , 11 ). DICER1 tumor predisposition syndrome (DTPS) is a genetic disorder that predisposes affected individuals to a wide range of tumors that have mainly pediatric onset (12)(13)(14). These lesions include non-toxic multinodular goiter (MNG), pleuropulmonary blastoma (PPB), cystic nephroma (CN), Sertoli-Leydig cell tumor (SLCT) of the ovaries, sarcomas arising at se v eral sites, and occasionally Wilms tumor (12)(13)(14). An individual is predisposed to such lesions if they harbor a germline pathogenic variant (GPV) in DICER1 . This variant is usually a nonsense or frameshift variant which is predicted to produce a truncated protein ( 12 ). Tumorigenesis then r equir es a second somatic variant (second hit) affecting one of two exons that encode the RNase IIIb domain of DICER1 in the originating tissue ( 15 ). Interestingly, a small subset of patients with DTPSrela ted clinical manifesta tions bear a germline DICER1 missense variant ( 12 ), se v eral of which fall into exons coding for the DICER1 Platform and PAZ domains. It can be difficult to determine if these variants are the cause of the syndromic presentations. This is because for tumor suppressor genes where the main pathogenic mechanism is biallelic inactivation of the causal gene in the associated tumors, it is expected that most causal variants will be predicted to truncate the protein. In this situation, the effect of missense variants on protein function is difficult to predict and therefore these variants are problematic for clinicians to interpret, leading to challenges to appropriate patient management ( 16 , 17 ).
Here, we utilized se v eral in vitro biochemical and cellbased assays to determine if an array of germline missense variants that cluster in the DICER1 Platform domain impact DICER1 activity in such a way that is consistent with a causal role in DTPS.

Plasmids
For generation of DICER1 mutant plasmids, pQCXIB-1xFLAG-DICER1 was used as a template for site-directed mutagenesis (SDM) using overlapping primers. For generation of Renilla miRNA sensor plasmids, we cloned the new target sites by digesting the pCl-RL-6xWT plasmid by conventional molecular cloning techniques using Not1 and Xba1 restriction enzymes (ThermoFisher Scientific).
We then ligated the cut plasmid by using overlapping oligos as the insert.

Antibodies
Antibody against DICER1 was purchased from Bethyl, a gainst beta-tub ulin from Millipore-Sigma, a gainst GAPDH from Cell-Signalling, against hnRNP A1 from Abcam. All antibodies were used at a dilution of 1:1000.

Immunoprecipitation and in vitro cleavage assays
Stable HEK 293 cell lines over expr essing DICER1 mutants were generated by gamma-retroviral infection produced by HEK293T cells that were transfected with pQCXIB-1xFLAG-DICER1, pUMVC and pVSVG plasmids. Stable cell lines were then grown to confluency in a 15 cm dish and pelleted. Frozen pellets were then lysed with 500ul of NP-40 lysis buffer (20 mM Tris-HCl, pH 7.6, 1mM EDTA, 150 mM NaCl, 0.4% (v / v) NP-40 (Igepal CA630) and complete protease inhibitor (Roche)). Pellets were lysed at 4˚C for 1 h. Lysates were then centrifuged at max speed for 15 min. The pr e-clear ed lysate was then incubated with 60 l of FLAG-M2 resin (Sigma) or agarose beads (Sigma), as a negati v e control, O / N at 4˚C on a rotator. The following day, the resin w as w ashed twice with NP-40 lysis, twice with IP wash buffer (20 mM Tris-HCl, pH 7.6, 1 mM EDTA, 300 mM NaCl, 0.4% (v / v) NP-40), and twice with DICER1 cleavage buffer (20 mM Tris-HCl, pH 7.6, 50 mM NaCl, 20% glycer ol, pr otein inhibitor (1:7)). Finally, DICER1 was eluted with 0.5 g / l of 3 × FLAG peptide (Sigma) in DICER1 cleava ge b uf fer. In vitr o cleavage assays wer e pr eviously described ( 18 ), which allows recognition of the pre-miRNA, and 5p / 3p miRNA cleavage products on the same gel as the 5p miRNA is designed to be longer than the 3p miRNA. To generate pre-miR-122, a three primer PCR was done using pre-miR-122 primers. The PCR product was then purified using the QIAquick Gel Extraction kit (Qiagen) as per manufactur er's protocol. Pr e-miRNA was transcribed via T7 polymerase with the MAXI-script kit using 200 ng of pre-miR-122 template and 6 l of radioacti v e UTP ([ ␣ ( 32 ) P]-800 ci / mmol) (PerkinElmer). The reaction was incuba ted a t 37˚C for 2 h. Then 1 l of DNase was added to the reaction that was then incubated at 37˚C for one extra hour. For each variant tested, immunoprecipitated FLAG-DICER1 was incubated with radiolabeled pre-miRNA in DICER1 cleava ge b uffer (see above) and 25 mM MgCl 2 , 20 mM DTT, and 0.25 l of RNase inhibitor (TakaraBio) for a total volume of 20 l. The reaction was incuba ted a t 37˚C and stopped at different intervals, including at the beginning of the reaction, after 1, 2 and 3 h by the addition of 20 l of RNA gel loading dye (Thermo Fisher Scientific) and then placed on ice.
Cleavage products were resolved on a 7 M urea, 15% polyacrylamide gel (19:1; acrylamide: bisacrylamide) dissolved in 1 × tris-borate-EDTA (TBE). The gels were then wrapped in saran wrap and exposed to a phosphor screen for 24 h a t 4˚C . The screen was then imaged by a phosphorimager (GE Storm 840, GE Healthcare or Amersham TYPHOON, Cytiva). Quantification of images was done by calculating the percent of cleaved product as previously described for DICER1 cleavage assays using ImageJ ( 19 ). NAR Cancer, 2023, Vol. 5, No. 3 3

Competition assays
Conditions were the same as for the in vitro cleavage assay except that, in addition to 50 ng of WT DICER1 protein, 50, 100 and 300 ng of competing protein was added to the reactions. Also, the cleavage reactions were only incubated at 37˚C for 1 h.

EMSA
Pr e-let-7i, and pr e-miR-100 wer e generated the same way as pre-miR-122 was for the in vitro cleavage reaction, with the exception that different primers were used for generation of the pre-let-7i and pr-miR-100 templates. 20 ng of radiolabelled RNA was then added with increasing amounts of DICER1 protein (0, 50, 100, 300 ng) in a binding solution consisting of 1 mM EDTA, 10 mM DTT, 20 mM Tris, 20% glycerol and 4.375 l of of Recombinant RNase Inhibitor (Takara) for a total volume of 35 l. The mixture was then incubated on ice for 30 min. EDTA was added to the reaction to pre v ent DICER1 from cleaving the pre-miRNA while still allowing binding ( 20 ). 5 l of EMSA loading buffer (20 mM tris, 50% glycerol, 0.03% bromophenol blue, 1 mM EDTA) was then added to the binding mixture and 30 l of the resulting solution was loaded on a nati v e 5% polyacrylamide TBE gel (29:1; acrylamide:bisacrylamide). The gel was then wrapped in saran wrap and exposed to a phosphor screen for 24 h a t 4˚C . The screen was then imaged by a phosphorimager (Amersham TYPHOON, Cytiva) and quantified using ImageJ.

MiRNA profiling
DICER1 knock-in mMSCs were generated following the same protocol used for infection of HEK 293 cells. Following one week of selection in 5 g / ml of blasticidin containing alpha-MEM media, the cells were single sorted in a 96well plate and expanded in 10 cm plates. Cells were allowed to expand between 90 and 100% confluency in 15 cm dishes. Cells were then directly lysed in the 15cm dish using the mir-Vana miRNA isolation kit (ThermoFisher Scientific) as per manufacturer's protocol. The miRNA was then diluted between 300 and 500 ng / l. Murine miRNAs were measured using the NanoString nCounter analysis system as per manufacturer's protocol.

Dual luciferase assays
Cells grown to 70% confluency in a 6-well plate were cotransfected with 20 ng of firefly plasmid and 20 ng of Renilla plasmid. Twenty-four hours post-transfection, the cells were harvested and lysed in Passi v e Lysis Buffer (Promega). The acti vity le v els of the Renilla and firefly reporters were measuring using Dual-Luciferase Assay (Promega).

DN A e xtraction and sequencing of DICER1 hotspot mutation
The multinodular goiter formalin-fixed paraffin embedded (FFPE) block was sliced at 10 m thickness and microdissected with a surgical blade based on the pathologist review of the biopsy. The scraped tissue was used for DNA extraction using the QIAmp DNA FFPE Tissue kit (Qiagen) as per manufacturer's protocol. DNA was amplified using a series of primers designed to amplify DICER1 hotspots for FFPE DNA. If bands were observed, the resulting PCR was sent for Sanger sequencing by Genome Qu ébec using the same primers that were used in the PCR.
DN A e xtraction from patient saliva / blood and germline sequencing 5 ml of patient blood or 4 ml of patient saliva was used for germline sequencing. DNA was then extracted using the Gentra Puregene DNA kit (Qiagen) as per manufacturer's protocol for either blood or saliva. DNA was amplified and sequenced with the same protocol as for FFPE DNA.
RN A e xtraction from patient blood and reverse-transcription-PCR (RT-PCR) 5 ml of patient blood was drawn and stored in EDTA-tubes. 3 ml was then used to extract RNA using the RiboPure Blood kit (Thermo Fisher Scientific) as per manufacturer's protocol. For cDNA generation, 1 g of purified RNA was used with 200 ng of random hexamer primers, with 1 l of 10 mM dNTP mix for a total volume of 13 l. The mixture was then incubated at 65˚C for 5 minutes and placed on ice for at least 1 minute. 4 l of 5 × First strand buffer, 1 l of 0.1M DTT, 1 l of RNase OUT Recombinant RNase Inhibitor, and 1 l of Superscript IV Re v erse Transcriptase (Thermo Fisher Scientific) were then added to the mixture. The reaction was then incubated at 25 ˚C for 5 min, 50˚C for 60 min and then inactivated at 70˚C for 15 min. 2 l of the cDNA was then used in a PCR with the same protocol used to amplify FFPE DNA and the amplicons were sequenced using Sanger sequencing (Genome Qu ébec).

Structural analyses
The DICER1 Platform / PAZ crystal structure (PDB:4NH3) ( 8 ) was used to model the variants on PyMOL version 2.5.0. To create a hydrophobic model, residues leucine (L), valine (V), isoleucine (I), glycine (G), methionine (M), phenylalanine (F), alanine (A) were changed to red to depict hydrophobic amino acids and all other r esidues wer e changed to blue to show hydrophilic r esidues. Mutations wer e introduced to the structure using the Wizard Mutagenesis script.

Western blotting
Samples wer e pr epar ed with equal volume of 2 × Laemmli buffer (4% SDS, 20% glycerol, 120 mM Tris-Cl (pH 6.8)) and boiled at 95˚C for 5 minutes. Samples were then separated on an 8% polyacrylamide gel electrophoresis (PAGE) gel. The samples were migrated at 165 V for 2 h in running buffer (1 M glycine, 1% SDS, 0.1 M Tris-HCl). The protein was then transferred to a 0.45 m nitrocellulose membrane (BioRad) in transfer buffer (1 M glycine, 0.1 M Tris-HCl, 10% methanol) at 30 V and 4˚C O / N. After blocking the membranes with 5% milk + TBST, the membranes were left in primary antibod y O / N a t 4˚C . The f ollowing da y the membranes were washed and incubated with secondary antibody for 1 h at room temperature. The resulting membranes were visualized by being covered with enhanced chemiluminescence substrate (BioRad). The membranes were placed in a plastic slee v e and visualized with ImageQuant LAS 4000 biomolecular imager (GE Healthcare).

Data analysis
Quantification of autor adiogr aphy images was done using ImageJ 1.53K, NIH, USA.

Statistical analyses
For analysis of in vitro cleavage assay and luciferase assay, P -values were calculated by one-way or two-way ANOVA using Dunnett's method with Prism version 8.4.0. All graphs wer e cr eated using Prism version 8.4.0. For miRNA profiling, normalization of miRNA reads was done using nSolver analysis software version 4 and as suggested by NanoString consultants by initially excluding miRNA reads in which the max read count across samples were less than 50 and then normalizing against miRNAs in which the coefficient variance (CV) was less than 20% that of the lowest miRNAs measured. Fold change was then measured compared to the human WT DICER1 knock in cell line. Volcano plots were generated by ROSALIND ® ( https:// rosalind.bio/ ), with a HyperScale ar chitectur e de v eloped by ROSALIND, Inc. (San Diego, CA). The limma R library ( 21 ) was used to calculate fold changes. T -test was used for P -value calculations with the Benjamini-Hochberg methods for controlling false discovery rate.

Identifying a cluster of germline missense variants that may associate with DTPS
We set out to gather data from the published literature on 18 germline DICER1 missense variants seen in persons with clinicopa thological manifesta tions tha t were mainly concordant with DTPS (Table 1 ). Within this list, we identified a cluster of missense variants that occurred in the Platform domain of DICER1 (Figure 1 A). To focus our efforts on variants that had a strong possibility of being diseaser elated, we filter ed this list by only retaining variants with a population allele frequency in the g e nom e a ggregation d atabase (gnomAD) under 0.01%, and that were predicted as damaging by at least two out of three in silico predictors: PolyPhen2, SIFT and Missense 3D ( 22 , 23 ). This analysis was done on all 18 variants and ultimately culminated with a list of four variants that clustered in the Platform domain (p.G803R, p.L805P, p.S839F and p.L881P), 1 in the RNase IIIa domain (p.L1583R), and 3 in the RNase IIIb domain (p .G1708E, p .L1777H, p .S1814L) (Figure 1 A, Tab le 1 ). Gi v en the unknown role of the Platform in DTPS, we decided to focus on variants in this domain. Based on our own laboratory data, we had previously indicated that we considered the variants p.G803R, p.L805P and p.L881P to be Likel y Patho genic, but we lacked further tumor sequencing information from persons with p.G803R and p .L881P germline variants . Additionally, from the variants in Table 1 , three out of six cases in which second soma tic DICER1 muta tions were identified in tumor DNA occurred in patients bearing germline missense variants in the Platform domain.
Sanger sequencing of DNA extracted from formalinfixed paraffin embedded (FFPE) thyroid nodules from individual V-8, (Figure 1 B, Table 2  Taken together with previously available tumor sequencing data for p. S839F ( 24 ) and p. L805P ( 25 ) these results strongly suggest that these four Platfor m ger mline variants are causal for DTPS-related tumors.

Select DICER1 platform mutants display impaired pre-miRNA cleavage capacity in vitro
Gi v en that the segregation and tumor sequencing data reported above lends credence to the notion that these variants are clinically important, it is interesting that an analysis of the DICER1 Platform domain crystal structure (Figure 2 A, B) re v ealed that p .G803, p .L805 and p .L881, are located near each other in a hydrophobic core (with p.S839 being close to this core) but not in the 5 phosphate binding pocket (Figure 2 C). Ne v ertheless, based on these data we hypothesized that germline variants in these residues may impact DICER1 activity. To test this, we carried out in vitro cleavage experiments with various DICER1 mutants. Briefly, a human pre-miR-122 (Figure 2 D) internally labelled with ( 32 P-UTP, was incubated with FLAG-tagged wild-type or mutant DICER1 proteins that were expressed and immunopurified from HEK-293 with FLAG antibody (Supplementary Figure S2). Pre-miR-122 cleavage by each FLAG-tagged DICER1 protein was then assessed by denaturing polyacrylamide gel electrophoresis (PAGE) followed by autor adiogr aphy. Wild-type DICER1 efficiently cleaved the miRNA precursor, leading to the production of both 5p and 3p miRNAs after 1 h of incubation (Figures 2 E and F). In contrast, two DICER1 germline Platform mutants, p .G803R and p .L805P, failed to cleave pre-miR-122 Table 1 Figure 2 G and H). Taken together, these results suggest that se v eral patientderi v ed DICER1 Platform domain variants fail to cleave a pre-miRNA in vitro .

Platf orm DICER1 v ariants fail to support miRN A e xpression and miRNA-mediated gene silencing in cells
Our in vitro cleavage assays suggest that these germline DICER1 variants inhibit miRNA processing. To determine the extent of these defects, we stably complemented  Figure S3). To determine whether alterations in miRNA le v els associated with different germline DICER1 variants impacts miRNA-mediated gene silencing, we transfected cells with plasmids encoding a Renilla luciferase (RL) reporter mRNA with six let-7 miRNA target sites (RL-6xB) or six mutated let-7 sites (RL-6xBMUT) in its 3 'UTR (Figure 4 A). 48 hours posttransfection, cells were lysed and RL-6xB silencing was assessed using luciferase assays, with RL-6xBMUT serving as a control. RL-6xB was efficiently silenced in Dicer1 −/ − MSCs complemented with exogenous DICER1 WT (Figure 4 B). In contrast, Dicer1 −/ − MSCs complemented with DICER1 G803R or DICER1 L805P displayed significant defects in silencing the RL-6xB reporter mRNA as compared to Dicer1 −/ − MSCs complemented with DICER1 WT (Figure  4 B). Similar r esults wer e observed when using other Renilla luciferase reporter mRNAs harboring single perfectly  complementary sites to a number of different differentially expressed miRNAs, including mmu-miR-29b, mmur-miR-148a, and mmu-miR-15b (Figures 4 C and D). Taken together, these data indicate that germline DICER1 Platform domain variants significantly impact miRNA expression, and as a result fail to establish miRNA-mediated gene silencing in cells.

Platform domain variants prevent DICER1 from binding to pre-miRNAs
The in vitro cleavage and miRNA profiling experiments suggested that se v eral ger mline Platfor m domain variants render DICER1 unable to process pre-miRNAs. This could be due to these variants directly impacting the activity of the DICER1 RNase III domains such that they are not able to cleave pre-miRNAs. However, another plausible model would be that these variants render DICER1 un-able to bind pre-miRNAs stem-loops (Figure 5 A). In keeping with the latter model, adding increasing amounts of either DICER1 G803R or DICER1 L805P Platform domain variants failed to pre v ent DICER1 WT fr om pr ocessing a radiolabeled pr e-miR-122 (Figur e 5 B). This is in contrast to competition assays whereby adding increasing amounts of an RNase-inacti v e DICER1 m utant, w hich harbors points mutations in both RNase IIIa (D1320A) and IIIb (E1813K) domains, gradually diminished the ability of DICER1 WT to generate 5p and 3p miRNAs (Figure 5 B). Similarly, adding increasing amounts of an RNase IIIa-or IIIbdead DICER1 variants led to a decrease in the ability of DICER1 WT to genera te ma tur e 3p and 5p miRNAs, r especti v ely (Figure 5 B).
To directly test if DICER1 Platform domain variants are defecti v e in pre-miRNA binding we ne xt carried out electrophoretic mobility shift assays (EMSAs). Briefly, constant amounts of different radiolabelled pre-miRNAs  (i.e. pr e-miR-122, pr e -let-7i and pre-miR-100) were titrated with increasing amounts of immunopurified DICER1 WT or select Platform domain variants. Following their incubation, RNA-protein complex es wer e r esolv ed by nati v e polyacrylamide gel electrophoresis (PAGE) and analyzed using a phosphorimager (Figures 6 A and B). While DICER1 WT efficiently bound all pre-miRNAs tested, DICER1 G803R and DICER1 L805P failed to bind to pre-miRNA stem-loops. Moreover, other Platform domain variants (p.S839F and p.L881P) that were partially defecti v e in generating mature miRNAs in vitro and in cells, failed to efficiently bind to pre-miRNAs in vitro . Taken together, these data indicate that ger mline DICER1 Platfor m domain variants are defecti v e in pre-miRNA processing due to their inabilities to bind to pre-miRNA stem-loops.

DISCUSSION
In this report, we have re vie wed data from 18 germline missense DICER1 variants and have identified four variants that are causal for DTPS. Using structural modeling, cell-free and cell-based assays we demonstrate that these missense variants structurally localize within the Platform domain and functionally behave as atypical loss of function variants.
The DICER1 Platform domain contains a 5 -recognition pocket that is r equir ed for DICER1 to recognize the pre-miRN A 5 -phosphate, with m utations in this basic motif reducing both cleavage efficiency and cleavage sites ( 7 , 8 ). We report here on se v eral germline DTPS-associated Platform domain variants that are defecti v e in pre-miRNA processing. Intriguingly, these variants do not reside within the Platform domain 5 -phosphate pocket. Molecular modeling suggests that the p.G803R variant introduces a long basic side chain that would clash with nearby residues L770, A832 and L881 (Figure 7 ). In addition, the p.L805P and p.L881P variants, which introduce a proline in the middle of their beta sheets may alter the overall Platform domain structure by disrupting the hydrogen-bonding network. In keeping with this, while p.L881P impaired DICER1 activity, the germline p.L881V variant, which should still form hydrogen bonds similar to L881, did not affect DICER1 cleavage in vitro (data not shown). Ne v ertheless, e xactly how these variants alter the DICER1 structure remains to be established.
Recently, the structural basis for DICER1-mediated pre-miRNA cleavage has been further elucidated. It was shown that the PAZ domain is reoriented to accommodate RNA for cleavage and that the Platform domain not only recognizes the 5 phosphate of pre-miRNA, but also plays a role in determining the cleavage pattern of pre-miRNA based on their 5 terminal base ( 11 ). Perhaps the flexibility of the PAZ domain may be impaired by these variants which in turn could explain the decreased substrate binding. Furthermore, it has been recently found that the recurrent DICER1 RNase IIIa variant (S1344L) impairs pre-miRNA cleavage similarly to somatic RNase IIIb variants by decreasing 5p miRNA le v els ( 26 , 27 ). Interestingly, no change in 5p to 3p miRNA ratio has been seen with Platform variants, indicating a different mechanism of action in disrupting miRNA biogenesis.
Most tumor suppressor genes associated with inherited cancer syndromes (e.g. retinoblastoma ( 28 )) obey Knudson's two-hit rule (Knudson, 1993), whereby both the first (germline) PV and second (tumor-confined) hit usually result in loss of function of the affected allele, Figure 8 A). DTPS is unusual because although the syndrome follows this two-hit model, the two hits are mechanistically distinct. In tumors occurring in DTPS, the first hit in DICER1 is generally a protein-truncating nonsense variant, but the second hit is nearly always a missense variant affecting DICER1 RNase IIIb activity ( 12 ) (Figure 8 B). Here, we describe a distinct DTPS phenomenon where the first hit is a mis-sense rather than a nonsense variant (Figure 8 C). Howe v er, as these missense variants in the Platform domain render DICER1 unable to bind pre-miRNA stem-loops, they are highl y likel y to be disease-associated alleles, and if occurring in a person with a tumor harboring a canonical RNase IIIb somatic mutant in trans , would be causal for DTPS. Ne v ertheless, it is generally held that in vitr o da ta pertaining to the behavior of germline variants should not be used by itself as the basis of clinical decision-making ( 29 ). Indeed, despite the compelling data presented here, supported by recently published data studying some the same variants ( 30 ), when we applied rules from the American College of Medical Genetics (with specifications from the DICER1 Variant Classification Expert Panel(VCEP) https://clinicalgenome. org/af filia tion/50050/ ) in an attempt to re-classify these v ariants, the v ariants with clear defects in pre-miRNA stem-loop binding (p.G803R and p.L805P) remained classified as variants of uncertain significance (VUS) ( Table  3 ). Ther efor e, until such time as the results of the binding and competition assays reported here are incorporated into DICER1 variant classification rules, the results presented here can provide clinicians with a greater degree of   confidence when interpreting rare variants in the Platform domain of DICER1 .

DA T A A V AILABILITY
The data underlying this article are available in the article and in its online supplementary material.